1. Field of the Invention
The present invention relates to a method for cloning and expressing a target gene by homologous recombination, and more particularly to a method for cloning and expressing a target gene by homologous recombination, wherein a host cell transformed with a recombinant vector and a plasmid containing a recombinase system is introduced with a linear DNA fragment which contains a target gene and a sequence having homology to the recombinant vector.
2. Background of the Related Art
DNA cloning refers to a technique for making a large amount of same gene family by linking a target gene to a self-replicable vector, such as plasmid, phage or cosmid, and introducing the vector into a host such as E. coli to proliferate the target gene. Cloning and sub-cloning in E. coli is performed through a method comprising linking a target gene amplified by a technique such as polymerase chain reaction (PCR) to a vector having a replication origin and an antibiotic selection marker using DNA ligase, introducing the resulting vector into E. coli cells, examining the antibiotic resistance of the bacteria and selecting the cloned cells.
This general cloning technique forms the basis of modern bioengineering and is widely used in the cloning and high-speed protein expression of mass amount of genes based on genetic information which has explosively increased after the human genome project accomplished. However, in the prior genetic engineering technique, there are limitations in that inserted DNA and a vector should be cut with a restriction enzyme at the same recognition site, and the restriction enzyme should be selected from those which do not cut the inside of the insertion DNA or vector. Also, the process of treating the inserted DNA and the vector with the restriction enzyme and ligase requires a skill, and much cost is incurred due to expensive enzymes.
Since the year 1990, interest in a genetic engineering technique utilizing sequence-specific recombinase, which recognizes a specific base sequence to promote the recombination between DNA fragments, has increased. Particularly, a Gateway system (Invitrogen Co.) for cloning genes using integrase that recognizes specific DNA sequences, such as attL, attR, attB and attP, has been developed and widely used in the rapid cloning and protein expression of mass amount of genes. This technique is performed by in vitro genetic engineering in the same manner as the conventional restriction enzyme-ligation reaction, but is a novel cloning method that utilizes LR clonase linking linear DNA fragments with attL or attR, and BP clonase mediating the recombination between attP and attB. In this method, a target gene can be cloned into an entry vector by conventional genetic engineering, and then transferred into various expression vectors by homologous recombination. Thus, this method is highly advantageous for the rapid sub-cloning and expression verification of mass amount of genes.
Meanwhile, if DNA fragments showing sequences identical at more than several hundreds of bases are present in E. coli cells, homologous recombination can occur due to recA and recBCD of E. coli itself. However, if homologous DNAs are less than 40-50 bases in length, genetic recombination by E. coli itself will not occur, and only if phage-derived recombinase Redα/β or RecE/T system is introduced, the recombination between homologous genes will occur. This genetic recombination utilizing short-length homologous DNA fragments can be used in a technique for manipulating microbial genomes, or in vivo cloning which is not affected by restriction enzymes/ligases (Zhang et al., Nature Genetics, 20:123, 1998, Zhang et al., Nature Biotechnology, 18:1314, 2000).
To perform in vivo cloning by the homologous recombination between a linearized plasmid vector and a PCR product, a method was used wherein homologous DNA of 40-50 bases in length is placed at both ends of inserted DNA, a vector is treated with restriction enzyme such that the homologous DNA region are also placed at both ends of the vector, and the vector is introduced into E. coli containing homologous recombinase. In this case, because the linearized cloning vector loses the replication ability of a circular plasmid, and thus does not show antibiotic resistance, the use of antibiotic resistance allows the determination of whether in vivo cloning is successful or not. Although this method is characterized by using recombinase in vivo in place of restriction enzymes or ligases, it has a shortcoming in that the linearized vector consisting of homologous DNA should also be constructed and used.
Proteomics, protein chips, structural biology and the like, which have recently been rapidly advanced, show a need for a genetic engineering technique that can easily clone mass amount of genes and rapidly express a target protein. Particularly, because it is very difficult to perform the existing cloning technique requiring a high degree of skill in an automatic and high-speed manner, there is a need for the development of an easy and rapid gene cloning and protein expression technique.
The present inventors have conducted studies to solve the above-described problems and, as a result, found that, if a target gene fragment is prepared to include sequences having homology to the forward ribosome recognition site and a portion of the region downstream of the promoter of a recombinant vector and to the backward ribosome recognition sequence and start codon of a first selection marker gene and is introduced into a host cell transformed with a plasmid, containing a recombinase system, and the recombinant vector, containing the first selection marker gene having deletions of the ribosome recognition sequence and the start codon, easy cloning of a target gene and the high-speed expression of protein can be achieved by homologous recombination without needing complicated genetic engineering steps, such as the restriction enzyme treatment and ligation of the vector and the inserted fragment, thereby completing the present invention.